Electron donor and acceptor utilization by halorespiring bacteria

Chlorinated ethenes are widespread soil and groundwater pollutants. Over the last 2 decades a lot of effort has been made to understand the degradation mechanisms for these pollutants. In the early eighties reduction of tetrachloroethene (PCE) was observed in anaerobic soil samples, which was shown to be mediated by microorganisms. The first microorganism able to couple the anaerobic reduction of PCE to growth in a process called halorespiration (alternative terms are chlororespiration, chloridogenesis or dehalorespiration) was isolated in 1993. Since then, about 15 bacteria able to reduce PCE metabolically have been isolated. This thesis describes research on different aspects influencing the reductive dechlorination of chlorinated ethenes by anaerobic halorespiring bacteria.

A new halorespiring bacterium is described in chapter 1. This bacterium, Sulfurospirillum halorespirans PCE-M2, was isolated from a polluted soil near Rotterdam harbor. Strain PCE-M2 is a metabolically versatile bacterium able to use a variety of electron acceptors and electron donors. This new strain is closely related to Dehalospirillum multivorans, but more detailed studies indicated that strain PCE-M2 belongs to the genus Sulfurospirillum, It also appeared that Dehalospirillum multivorans had to be included in this genus. Consequently, it was reclassified to Sulfurospirillum multivorans.

Members of the genus Sulfurospirillum were originally known for their sulphur, selenate and arsenate respiring properties. Therefore, we screened a number of halorespiring and related bacteria for their metal reducing properties (Chapter 2). It was shown that the reduction of metals such as ferric iron, manganese, selenate and arsenate is a common property amongst halorespiring bacteria. We also investigated the quinone reducing and oxidizing abilities. AU tested bacteria are able to reduce AQDS7 a quinone-bearing humic acid analogue. Some of the tested bacteria (Desulfitobacterium hafniense DP7, Sulfurospirillum barnesii, S. deleyianum and S. arsenophilum) are also able to oxidize AEbQDS coupled to nitrate reduction.

The influence of some alternative electron acceptors on the reductive dechlorination is discussed in chapter 3. Sulfurospirillum halorespirans preferably reduces nitrate (to ammonium) and then PCE. In contrast, Sulfurospirillum multivorans reduces nitrate only to nitrite, and PCE reduction is blocked irreversibly in the presence of nitrate. In Desulfitobacterium frappieri TCEl, PCE and nitrate are reduced simultaneously in excess of electron donor. Under electron donor limitation PCE reduction was inhibited (Gerritse Bt al., AppI. Environ. Microbiol. 1999, 65, 5212-5221). The influence of nitrate on the reduction of chlorinated ethenes by halorespiring bacteria differs between species and may also depend on the availability of electron donor. Sulphate, which is not used as electron acceptor by chlorinated ethenes respiring bacteria is often found at polluted sites. We have tested the influence of sulphate on halorespiring bacteria (Chapter 3). It appeared that sulphate does not influence these microorganisms. Sulphite however, a possible electron acceptor for Desulfitobacterium species, inhibits the reduction of PCE. This inhibition may be the result of a chemical interaction between sulphite and cobalamine containing dehalogenases. We also studied the adaptation of Sulfurospirillum halorespirans PCE-M2 to different alternative electron acceptors (Chapter 3). Both nitrate and arsenate are reduced by cells pre-grown on PCE, nitrate, arsenate and selenate. This indicates that the enzymes responsible for the reduction of nitrate and arsenate are constitutiveiy present in S, halorespirans. In contrast, PCE and selenate are only reduced by cells pre-grown on PCE or selenate respectively.

Halorespiring bacteria have a high affinity for hydrogen (H2). H2 may even be the most important electron donor for these organisms in natural environments. We have studied H2^thresh0ld concentrations in pure cultures of halorespiring bacteria (Chapter 4). H2-threshold values between 0.05 and 0.08 nM under PCE-reducing and nitrate-reducing conditions were measured. Furthermore, we measured H2 concentrations at a field site polluted with chlorinated ethenes. PCE and trichloroethene (TCE) reduction can occur at H2 concentrations below 1 nM. However, for the reduction of lower chlorinated ethenes a higher H2 concentration seems to be required.

Accumulation of cis-l,2-dichloroethene (cis-l,2-DCE) and vinyl chloride (VC) under anaerobic conditions is often observed. The enrichment of two cultures (DCE-I and DCE-2) able to reduce VC at relative high rates is described in chapter 5. Cis-l,2-DCE is reduced at approximately 20-30 fold lower rates than VC. Our results suggest that these two enrichment cultures are able to gain energy from the reduction of lower chlorinated ethenes. When we performed these studies, no microorganisms had been isolated able to grow by the reduction of VC. However, recently He et al. (Nature. 2003, 424, 62-65) isolated Dehalococcoides strain BAVl, which is able to couple the reduction of DCE and VC to ethene to growth.

Finally, the results obtained are combined with available literature data to obtain a state-of-the-art on chlorinated ethenes respiring microorganisms, the influence of alternative electron acceptors on these microorganisms and the role of H3 and H?-threshold values in halorespiration.